Historical and Current Concepts of Fibrillogenesis and In vivo Amyloidogenesis: Implications of Amyloid Tissue Targeting.

Historical and current concepts of in vitro fibrillogenesis are considered in the light of disorders in which amyloid is deposited at anatomic sites remote from the site of synthesis of the corresponding precursor protein. These clinical conditions set constraints on the interpretation of information derived from in vitro fibrillogenesis studies. They suggest that in addition to kinetic and thermodynamic factors identified in vitro, fibrillogenesis in vivo is determined by site specific factors most of which have yet to be identified.

Heparan sulfate dissociates serum amyloid A (SAA) from acute-phase high-density lipoprotein, promoting SAA aggregation.

Inflammation-related (AA) amyloidosis is a severe clinical disorder characterized by the systemic deposition of the acute-phase reactant serum amyloid A (SAA). SAA is normally associated with the high-density lipoprotein (HDL) fraction in plasma, but under yet unclear circumstances, the apolipoprotein is converted into amyloid fibrils. AA amyloid and heparan sulfate (HS) display an intimate relationship in situ, suggesting a role for HS in the pathogenic process. This study reports that HS dissociates SAA from HDLs isolated from inflamed mouse plasma. Application of surface plasmon resonance spectroscopy and molecular modeling suggests that HS simultaneously binds to two apolipoproteins of HDL, SAA and ApoA-I, and thereby induce SAA dissociation. The activity requires a minimum chain length of 12-14 sugar units, proposing an explanation to previous findings that short HS fragments preclude AA amyloidosis. The results address the initial events in the pathogenesis of AA amyloidosis.

Acute-phase serum amyloid A: perspectives on its physiological and pathological roles.

Serum amyloid A (SAA), a protein originally of interest primarily to investigators focusing on AA amyloidogenesis, has become a subject of interest to a very broad research community. SAA is still a major amyloid research topic because AA amyloid, for which SAA is the precursor, is the prototypic model of in vivo amyloidogenesis and much that has been learned with this model has been applicable to much more common clinical types of amyloid. However, SAA has also become a subject of considerable interest to those studying (i) the synthesis and regulation of acute phase proteins, of which SAA is a prime example, (ii) the role that SAA plays in tissue injury and inflammation, a situation in which the plasma concentration of SAA may increase a 1000-fold, (iii) the influence that SAA has on HDL structure and function, because during inflammation the majority of SAA is an apolipoprotein of HDL, (iv) the influence that SAA may have on HDL's role in reverse cholesterol transport, and therefore, (v) SAA's potential role in atherogenesis. However, no physiological role for SAA, among many proposed, has been widely accepted. None the less from an evolutionary perspective SAA must have a critical physiological function conferring survival-value because SAA genes have existed for at least 500 million years and SAA's amino acid sequence has been substantially conserved. An examination of the published literature over the last 40 years reveals a great deal of conflicting data and interpretation. Using SAA's conserved amino acid sequence and the physiological effects it has while in its native structure, namely an HDL apolipoprotein, we argue that much of the confounding data and interpretation relates to experimental pitfalls not appreciated when working with SAA, a failure to appreciate the value of physiologic studies done in the 1970-1990 and a current major focus on putative roles of SAA in atherogenesis and chronic disease. When viewed from an evolutionary perspective, published data suggest that acute-phase SAA is part of a systemic response to injury to recycle and reuse cholesterol from destroyed and damaged cells. This is accomplished through SAA's targeted delivery of HDL to macrophages, and its suppression of ACAT, the enhancement of neutral cholesterol esterase and ABC transporters in macrophages. The recycling of cholesterol during serious injury, when dietary intake is restricted and there is an immediate and critical requirement of cholesterol in the generation of myriads of cells involved in inflammation and repair responses, is likely SAA's important survival role. Data implicating SAA in atherogenesis are not relevant to its evolutionary role. Furthermore, in apoE(-/-) mice, domains near the N- and C- termini of SAA inhibit the initiation and progression of aortic lipid lesions illustrating the conflicting nature of these two sets of data.

Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein.

Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.

Secretion of amyloidogenic gelsolin progressively compromises protein homeostasis leading to the intracellular aggregation of proteins.

Familial amyloidosis of Finnish type (FAF) is a systemic amyloid disease associated with the deposition of proteolytic fragments of mutant (D187N/Y) plasma gelsolin. We report a mouse model of FAF featuring a muscle-specific promoter to drive D187N gelsolin synthesis. This model recapitulates the aberrant endoproteolytic cascade and the aging-associated extracellular amyloid deposition of FAF. Amyloidogenesis is observed only in tissues synthesizing human D187N gelsolin, despite the presence of full-length D187N gelsolin and its 68-kDa cleavage product in blood-demonstrating the importance of local synthesis in FAF. Loss of muscle strength was progressive in homozygous D187N gelsolin mice. The presence of misfolding-prone D187N gelsolin appears to exacerbate the age-associated decline in cellular protein homeostasis (proteostasis), reflected by the intracellular deposition of numerous proteins, a characteristic of the most common degenerative muscle disease of aging humans, sporadic inclusion body myositis.

Heparan sulfate promotes the aggregation of HDL-associated serum amyloid A: evidence for a proamyloidogenic histidine molecular switch.

During inflammatory diseases, serum amyloid A (SAA), an acute-phase apolipoprotein of HDL, can assemble into tissue deposits called AA amyloids. The mechanism and physiological factors promoting amyloidosis are largely unknown but likely involve heparan sulfate (HS), a glycosaminoglycan colocalized with all types of amyloids. In this study, we explored HDL-SAA:HS interactions using in vitro and cell culture assays to identify HS-binding domains that promote the conversion of native SAA into AA amyloid. HS causes the remodeling of HDL-SAA at mildly acidic pH, producing SAA-rich aggregates. A sequence motif in SAA responsible for this conversion was identified that contains a pH-sensitive heparin/HS-binding site, functions as a ligand for a cell surface receptor, and acts as a structural focal point for SAA aggregation. Synthetic peptides corresponding to this region promoted the deposition of AA amyloid in a monocyte culture model for AA amyloidogenesis. The effects were peptide sequence specific and reliant on the protonation of H36. We conclude that a highly conserved motif required for SAA binding to macrophages can, under acidic pH conditions and in an HS-dependent manner, also act as a molecular switch, directing SAA misfolding into AA amyloid. Similar histidine-dependent HS-binding sites are also found in other amyloidogenic polypeptides.

Acute-phase-HDL remodeling by heparan sulfate generates a novel lipoprotein with exceptional cholesterol efflux activity from macrophages.

During episodes of acute-inflammation high-density lipoproteins (HDL), the carrier of so-called good cholesterol, experiences a major change in apolipoprotein composition and becomes acute-phase HDL (AP-HDL). This altered, but physiologically important, HDL has an increased binding affinity for macrophages that is dependent on cell surface heparan sulfate (HS). While exploring the properties of AP-HDLratioHS interactions we discovered that HS caused significant remodeling of AP-HDL. The physical nature of this change in structure and its potential importance for cholesterol efflux from cholesterol-loaded macrophages was therefore investigated. In the presence of heparin, or HS, AP-HDL solutions at pH 5.2 became turbid within minutes. Analysis by centrifugation and gel electrophoresis indicated that AP-HDL was remodeled generating novel lipid poor particles composed only of apolipoprotein AI, which we designate beta2. This remodeling is dependent on pH, glycosaminoglycan type, is promoted by Ca(2+) and is independent of protease or lipase activity. Compared to HDL and AP-HDL, remodeled AP-HDL (S-HDL-SAA), containing beta2 particles, demonstrated a 3-fold greater cholesterol efflux activity from cholesterol-loaded macrophage. Because the identified conditions causing this change in AP-HDL structure and function can exist physiologically at the surface of the macrophage, or in its endosomes, we postulate that AP-HDL contains latent functionalities that become apparent and active when it associates with macrophage cell surface/endosomal HS. In this way initial steps in the reverse cholesterol transport pathway are focused at sites of injury to mobilize cholesterol from macrophages that are actively participating in the phagocytosis of damaged membranes rich in cholesterol. The mechanism may also be of relevance to aspects of atherogenesis.

Serum amyloid A, in vivo splenic cholesterol export and its potential implications in hemolytic disorders.

A model to examine the in vivo relationship of acute phase serum amyloid A (SAA) to spleen cholesterol mobilisation was devised. Reticuloendothelial cells in vivo were loaded with a known quantity of cholesterol (1.5 mg) by infusing fragmented red blood cell membranes, which consist of approximately 50% cholesterol by dry weight. Following infusion, 7% of the infused cholesterol was in the spleen and significantly increased (by 35%) spleen cholesterol concentration above the baseline. An acute inflammatory reaction was induced by the subcutaneous injection of AgNO(3) which also raised spleen cholesterol values, but not significantly. Both treatments were also administered together and the increase in spleen cholesterol concentration after 1 h was equivalent to the sum of the individual treatments. In all the treatment groups, the spleen cholesterol concentration and the plasma SAA values were then followed over a period of 24 h. In all treatment groups the spleen cholesterol values fell to baseline values primarily between 18 and 24 h which coincided with significantly raised levels of plasma SAA. In the case of the dual treatment, between 4 and 18 h, SAA increased from 92.1 +/- 12.3 to 478 +/- 58.3 microg/ml, respectively and depletion of spleen cholesterol occurred gradually reaching baseline values after 24 h. The significant flux of cholesterol though the spleen raises the distinct possibility that the spleen is much more involved in cholesterol metabolism than previously appreciated. Furthermore, the speed with which plasma SAA increases following the infusion of fragmented red blood cell membranes and the role that SAA plays in cholesterol mobilisation raise issues that may be relevant to alterations in plasma acute phase protein and lipid parameters in patients undergoing transfusions or suffering from hemolytic disorders.

The anti-atherogenic potential of serum amyloid A peptides.

Serum amyloid A (SAA) inhibits acyl coenzyme A cholesterol acyltransferase and enhances cholesterol esterase activities, shifting stored esterified cholesterol to free cholesterol (the exportable form). The SAA domains responsible for these enzyme-modifying properties have been identified. These peptides are sufficiently small to be synthetically prepared to GMP quality and used in vivo to alter the progression of aortic lipid lesions in models of atherogenesis, suggesting that they may be clinically useful. The residues critical for peptide function are the foundation for corresponding small-molecule development. In association with SAA studies, a novel macrophage in vivo assay is described that monitors macrophage cholesterol efflux, as well as its utility in the study of anti-atherogenic compounds, and its adaptation for clinical use. A brief history of the role of SAA in amyloid A amyloidosis and its potential role in atherogenesis is included, along with a description of the function of SAA in mobilizing macrophage cholesterol for export.

Inhibition of glycosaminoglycan synthesis and protein glycosylation with WAS-406 and azaserine result in reduced islet amyloid formation in vitro.

Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in approximately 90% of individuals with Type 2 diabetes and is associated with decreased islet beta-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet beta-cells. In addition to IAPP, islet amyloid deposits contain other components, including heparan sulfate proteoglycans (HSPGs). The small molecule 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha-D-xylo-hexopyranose (WAS-406) inhibits HSPG synthesis in hepatocytes and blocks systemic amyloid A deposition in vivo. To determine whether WAS-406 inhibits localized amyloid formation in the islet, we incubated hIAPP transgenic mouse islets for up to 7 days in 16.7 mM glucose (conditions that result in amyloid deposition) plus increasing concentrations of the inhibitor. WAS-406 at doses of 0, 10, 100, and 1,000 microM resulted in a dose-dependent decrease in amyloid deposition (% islet area occupied by amyloid: 0.66 +/- 0.14%, 0.10 +/- 0.06%, 0.09 +/- 0.07%, and 0.004 +/- 0.003%, P < 0.001) and an increase in beta-cell area in hIAPP transgenic islets (55.0 +/- 2.6 vs. 60.6 +/- 2.2% islet area for 0 vs. 100 microM inhibitor, P = 0.05). Glycosaminoglycan, including heparan sulfate, synthesis was inhibited in both hIAPP transgenic and nontransgenic islets (the latter is a control that does not develop amyloid), while O-linked protein glycosylation was also decreased, and WAS-406 treatment tended to decrease islet viability in nontransgenic islets. Azaserine, an inhibitor of the rate-limiting step of the hexosamine biosynthesis pathway, replicated the effects of WAS-406, resulting in reduction of O-linked protein glycosylation and glycosaminoglycan synthesis and inhibition of islet amyloid formation. In summary, interventions that decrease both glycosaminoglycan synthesis and O-linked protein glycosylation are effective in reducing islet amyloid formation, but their utility as pharmacological agents may be limited due to adverse effects on the islet.

Heparan sulfate as a therapeutic target in amyloidogenesis: prospects and possible complications.

Amyloid formation in vivo is a much more complicated process than studies of in vitro protein/peptide fibrillogenesis would lead one to believe. Amyloidogenesis in vivo involves multiple components, some no less important than the amyloidogenic protein/peptides themselves, and each of these components, and its role in the pathogenetic steps toward amyloid deposition could, theoretically, be a therapeutic target. Herein we use the definition of amyloid as it was originally described, discuss the similarities and differences between amyloid in vivo and in vitro, address the potential role of the extracellular matrix in in vivo amyloidogenesis by focusing on a specific component, namely heparan sulfate proteoglycan, and describe studies illustrating that heparan sulfate is a valid target for anti-amyloid therapy. In light of experimental and recent clinical results obtained from studies addressing heparan sulfate's role in amyloid deposition additional novel anti-amyloid therapeutic targets will be proposed. Lastly, given the multiple roles that heparan sulfate plays in organ development, and organ and cell function, potential side effects of targeting heparan sulfate biosynthesis for therapeutic purposes are considered.

Sulfated cyclodextrins inhibit the entry of Plasmodium into red blood cells. Implications for malarial therapy.

The effect of sulfated cyclodextrins on Plasmodium falciparum cultures was determined. alpha-, beta-, and gamma-Cyclodextrins having equal degrees of sulfation inhibited parasite viability to a similar degree, a result suggesting that the ring size of the cyclodextrin is not a critical factor for inhibitory activity. beta-Cyclodextrins containing fewer than two sulfate groups had no inhibitory activity, however, compounds containing 7-17 sulfates were found to be active in the microM range. Examination of treated cultures indicated that intracellular forms of the parasite were unaffected; however, increased numbers of extracellular merozoites were present. Active compounds produced enhanced erythrocyte staining with cationic dyes that could be reduced by stilbene disulfonates, a result suggesting that sulfated cyclodextrins inhibit parasite growth by interacting with the anion transport protein, AE1. Compounds that were found to be active in P. falciparum cultures were also found to inhibit P. berghei merozoite entry and could reduce the parasitemia of P. berghei infection in a mouse model, results suggesting that these compounds inhibit a common step in the merozoite invasion process of at least two Plasmodium species.

The path of murine serum amyloid A through peritoneal macrophages.

Serum amyloid A (SAA) is a family of proteins encoded by four related genes. Of the four, isoforms 1.1 and 2.1 are acute phase proteins synthesized by the liver. They become major components of the HDL plasma fraction during acute tissue injury and the HDL/SAA complex is readily taken up by macrophages. Herein we investigated the path SAA follows when presented to macrophages as HDL/SAA or in liposomes. Using antibodies specific to SAA and confocal microscopy, or EM autoradiography where only SAA is radio-labeled, we show that HDL/SAA is taken up rapidly by macrophages and within 30 min SAA, or fragments thereof, proceeds through the cytoplasm to the peri-nuclear region and then the nucleus. Within 45-60 min SAA, or fragments thereof, is found back in the cytoplasm and at the plasma membrane where it is subsequently extruded. The observation that SAA, or fragments thereof, traverse the nucleus is a novel finding and may implicate SAA in macrophage gene regulation. It also raises questions by what mechanism SAA enters and leaves the nucleus. We further investigated if both SAA isoforms traffic through the macrophage in a similar manner. Isoform differences were observed. Both isoforms bind well to the plasma membrane of macrophages at 4 degrees C, but at 37 degrees C only SAA2.1 is taken up by the cell in significant quantity, and is observed in the nucleus, suggesting that the two isoforms are handled differently and that they may have discrete physiological roles.

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro.

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[3H]glucosamine, [35S]sulfate, and L-[14C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-(3) and beta-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[3H]glucosamine, but not of [35S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[14C]leucine incorporation into total protein synthesis. These results suggest that analogs 3-5 exhibit an inhibitory effect on D-[3H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[3H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-alpha-(6) and beta-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[3H]glucosamine and [35S]sulfate incorporation into isolated GAGs, namely to approximately 7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (approximately 40 kDa) than for control cultures (approximately 77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D-[3H]glucosamine and [35S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.

Peptides derived from serum amyloid A prevent, and reverse, aortic lipid lesions in apoE-/- mice.

Macrophages (Mphi) at sites of acute tissue injury accumulate and export cholesterol quickly. This metabolic activity is likely dependent on the physiological function of a major acute-phase protein, serum amyloid A 2.1 (SAA2.1), that is synthesized by hepatocytes as part of a systemic response to acute injury. Our previous studies using cholesterol-laden J774 mouse Mphi showed that an N-terminal domain of SAA2.1 inhibits acyl-CoA:cholesterol acyltransferase activity, and a C-terminal domain enhances cholesteryl ester hydrolase activity. The net effect of this enzymatic regulation is to drive intracellular cholesterol to its unesterified state, the form readily exportable to an extracellular acceptor such as HDL. Here, we demonstrate that these domains from mouse SAA2.1, when delivered in liposomal formulation, are effective at preventing and reversing aortic lipid lesions in apolipoprotein E-deficient mice maintained on high-fat diets. Furthermore, mouse SAA peptides, in liposomal formulation, are effective at regulating cholesterol efflux in THP-1 human Mphi, and homologous domains from human SAA are effective in mouse J774 cells. These peptides operate at the level of the foam cell in the reverse cholesterol pathway and therefore may be used in conjunction with other agents that act more distally in this process. Such human peptides, or small molecule mimics of their structure, may prove to be potent antiatherogenic agents in humans.

Preparation and propagation of amyloid-enhancing factor.

Amyloid-enhancing factor (AEF) is a biological "activity" that is defined in the context of inflammation-associated amyloidogenesis (AA). When administered intravenously to mice followed by an inflammatory stimulus, such primed mice deposit substantial AA amyloid in spleen within 36-48 h. Since experimental induction of AEF is dependent on amyloidogenic protocols, and rapid AA amyloid induction is dependent on AEF, a strategy for AEF isolation is required to break into this circular process. AEF activity may be prepared from a variety of human forms of amyloid that include tissue containing any of Abeta, AA, ATTR, and AL amyloids. The preparation of an AEF extract from such human tissue is described using 4 M glycerol, which then may be used to induce splenic AA amyloid fibrils in mice as a source for the propagation of additional AEF and/or for the study of amyloidogenesis. The glycerol and AA fibril preparations are stable frozen for many years.

In vivo fragmentation of heparan sulfate by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis.

Amyloid diseases encompass >20 medical disorders that include amyloid protein A (AA) amyloidosis, Alzheimer's disease, and type 2 diabetes. A common feature of these conditions is the selective organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have generated transgenic mice that overexpress human heparanase and have tested their susceptibility to amyloid induction. Drastic shortening of heparan sulfate chains was observed in heparanase-overproducing organs, such as liver and kidney. These sites selectively escaped amyloid deposition on experimental induction of inflammation-associated AA amyloidosis, as verified by lack of material staining with Congo Red, as well as lack of associated polysaccharide, whereas the same tissues from control animals were heavily infiltrated with amyloid. By contrast, the spleens of transgenic mice that failed to significantly overexpress heparanase contained heparan sulfate chains similar in size to those of control spleen and remained susceptible to amyloid deposition. Our findings provide direct in vivo evidence that heparan sulfate is essential for the development of amyloid disease.

Amyloidogenesis recapitulated in cell culture: a peptide inhibitor provides direct evidence for the role of heparan sulfate and suggests a new treatment strategy.

To date 22 different polypeptides, including Abeta in Alzheimer's disease and PrP(Sc) in prion disorders, are known to re-fold and assemble into highly organized fibrils, which associate with heparan sulfate (HS) proteoglycans to form tissue deposits called amyloid. Mononuclear phagocytes have long been thought to be involved in this process, and we describe a monocytic cell culture system that can transform the acute-phase protein serum amyloid A (SAA1.1) into AA-amyloid and appears to recapitulate all the main features of amyloidogenesis observed in vivo. These features in common include nucleation-dependent kinetics, identical proteolytic processing of SAA1.1, and co-deposition of HS with the fibrils. Heparin and polyvinylsulfonate previously reported to block AA-amyloidogenesis in mice are also effective inhibitors in this cell culture model. Furthermore, a synthetic peptide (27-mer) corresponding to a HS binding site of SAA, blocks amyloid deposition at a concentration that is several-orders-of-magnitude lower than any other peptide-based inhibitor previously reported. The 27-mer's inhibitory activity may target the amyloidogenic pathway specifically as it does not interfere with the binding of SAA to monocytes. These data provide direct evidence that SAA1.1:HS interactions are a critical step in AA-amyloidogenesis and suggest a novel treatment strategy for other amyloidoses.

Novel glycosaminoglycan precursors as antiamyloid agents: Part IV.

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, beta-amyloid (Abeta), in Alzheimer's disease. The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan, part of which is protein and the remainder a specific linear polysaccharide, which is the portion responsible for binding and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. Similarly, 4-deoxy analogs of glucosamine (a precursor of HS biosynthesis) are effective antiamyloid compounds both in culture and in vivo. Our continuing work concerns (1) the testing of our 4-deoxy compounds in a mouse transgenic model of Alzheimer's disease, and (2) the continuing design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it affects its amyloid-inducing properties. Since our previous report, 22 additional compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Of these, 12 soluble compounds have been assessed for their effect on HS biosynthesis in hepatocyte tissue cultures. In addition, one anomer of a 4-deoxy-d-glucosamine analog, which possesses AA-amyloid inhibitory properties in vivo is in the process of being assessed for its anti-Abeta activity using a murine transgenic model of brain Abeta amyloidogenesis. The majority of the novel sugars prepared to date are analogs of N-acetylglucosamine. They have been modified at the 2-N, C-3, C-4, C-3 and C-4, or C-6 positions. One compound modified at the 2-N position (QS231), which inhibits HS synthesis in hepatocyte cultures, has shown marked enhancing properties vis-à-vis AA amyloid deposition in vivo. Very instructive results with regard to HS structure and its relation to AA amyloid deposition should be forthcoming from analyses of the AA-associated HS generated with this compound.

Inhibition of amyloid A amyloidogenesis in vivo and in tissue culture by 4-deoxy analogues of peracetylated 2-acetamido-2-deoxy-alpha- and beta-d-glucose: implications for the treatment of various amyloidoses.

Two novel sugars, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha- and beta-D-xylo-hexopyranoses, have been synthesized and their effects on heparan sulfate biosynthesis using primary mouse hepatocytes in tissue culture have been assessed. At concentrations of 0.1 and 1.0 mmol/L a mixture of both anomers significantly inhibited the biosynthesis of heparan sulfate by 60% and 99%, respectively. At 1.0 mmol/L the average molecular weight of the heparan sulfate synthesized is reduced from 77 kd to 40 kd. The biosynthetic inhibition is apparent within 1 hour (the earliest time point examined) of exposure of the hepatocytes to the analogues and appears virtually complete throughout a 24-hour incubation period. Using a radiolabeled version of the beta-anomer we demonstrate that the analogue is incorporated into growing heparan sulfate chains. The nature of the analogue, the quantity of analogue isotope incorporated, and the reduction in the size of the heparan sulfate polysaccharide are consistent with UDP activation and incorporation of the analogue and truncation of the growing heparan sulfate chain. At 0.1 mmol/L, and in the presence of a constant concentration of serum amyloid A (the precursor to AA amyloid), each analogue inhibited amyloid deposition (by 95 to 99%) in a tissue culture model of AA amyloidogenesis. At 6 mg/dose twice daily each analogue inhibited in vivo splenic AA amyloid deposition by 65 to 70% when using a rapid induction model of mouse AA amyloidogenesis. These data indicate that polysaccharides, such as heparan sulfate, play an integral part in the pathogenesis of AA amyloid deposition, and potentially other forms of amyloid. These data support our previous work that demonstrated that agents that mimic aspects of heparan sulfate structure and that interfere with heparan sulfate:amyloid protein binding inhibit AA amyloid deposition. They emphasize that heparan sulfate likely plays a critical role in amyloidogenesis, and compounds that interfere with heparan sulfate biosynthesis may provide leads for the development of anti-amyloid therapeutic agents.